Type 2 diabetes mellitus incidence has increased dramatically. Among the life threatening complications is heart failure, which is preceded by bioenergetic dysfunction. Using mouse (db/db) and human (patient) type 2 diabetic models, we observed pronounced mitochondrial dysfunction culminating in a decreased ability to generate ATP for cardiac contraction. MicroRNAs (miRs) are non-coding RNAs that regulate translation. Using cross-linking immunoprecipitation and deep sequencing, we made the exciting observation, in both db/db and type 2 diabetic patients that miRs translocate into and out of cardiac mitochondria. Of particular interest was an increased miR- 378 presence in a functional regulatory context with mitochondrial genome-encoded ATP6 mRNA which codes for a subunit of the F0 proton motor that is part of the ATP synthase complex. Decreased ATP synthase functionality promotes bioenergetic deficit in the heart, promoting heart failure. Nevertheless, it is currently unclear whether miR-378 blockade can reduce mitochondrial dysfunction associated with the type 2 diabetic heart by direct interaction with the mitochondrial transcriptome. Further, the mechanisms responsible for the dynamic flux of miRs into the mitochondrion are undefined. One potential mechanism involves the participation of the mitochondrial RNA import protein polynucleotide phosphorylase (PNPase) which we have observed to be increased in mitochondria from db/db mice and type 2 diabetic patients. The studies being proposed address these gaps in knowledge and integrate in vitro cellular approaches with animal and human experimental models in an effort to begin to translate the findings to the type 2 diabetic patient. The objectives of this application are (1) determine the efficacy in vivo of miR-378 loss or its functional inhibition in a type 2 diabetic mouse model for restoring mitochondrial ATP6 protein expression and ATP generating capacity in the heart; (2) evaluate the therapeutic efficacy of a miR-378 inhibitor delivered to isolated human cardiomyocytes from type 2 diabetic patients; and (3) assess the contribution of PNPase to the mechanisms driving miR-378 flux into the mitochondrion. The central hypothesis of this application is that inhibition of miR-378 will disrupt its ability to translationally down-regulate ATP6 in the mitochondrion, preserving ATP generating capacity and limiting cardiac contractile dysfunction in the type 2 diabetic heart. Further, miR-378 flux into the mitochondrion can be modulated by manipulating PNPase levels and its structure. To test this hypothesis, an innovative approach has been proposed which employs novel experimental methodologies that are tested in cellular, animal and human models. The combination of work proposed is significant because it will provide insight into the mechanisms regulating miR distribution in the mitochondrion while providing initial translational insight into the therapeutic potential of miR-378 inhibition as a treatment strategy. Our approach merges mechanistic examination of a previously unexplored regulatory pathway contributing to mitochondrial dysfunction in the type 2 diabetic heart with preclinical evaluation of key molecular constituents participating in the axis.